The determination of the MICs for antimicrobial activity requires a 24h exposure time, but for the cytotoxicity study, in the literature, I only find 1h exposure time for the hemolysis assay. Why not 24h exposure time for the hemolysis assay? Is a 1h hemolysis assay relevant when you want to study the cytotoxicity of a compound whose MIC was determined on a 24h period ?
The kinetics of cell lysis with complement verses microbial growth (or inhibition thereof) in the presence and absence of antimicrobial drug are different. The active components have different half lives and the readout times for reproducible results vary. For example C3 convertases are unstable (half-life 10 – 20 min at 37C) – respectively they are deactivated upon spontaneous dissociation or by facilitated dissociation mediated by the regulators of complement activation proteins decay acceleration factor (DAF), complement receptor 1 (CR1), C4b-binding protein and Factor H. Convertase assembly is suppressed by the proteolytic cleavage of C3b (and C4b) as mediated by Factor I in the presence of membrane cofactor protein (MCP, CD46), C4b-binding protein, CR1, or a plasma-glycoprotein Factor H. The hemolysis assay as recommended in the literature also requires complement titration and uses a one fold complement excess of what is required for 100% lysis within one hour. This is arbitrary and could just as easily be 30 minutes - 3 hours presuming complement is available throughout the incubation period and discrimination within the assay is achieved. In contrast the MIC requires microbial division to a level of discriminating unimpeded growth from drug impeded growth either by eye or electronically. Thus the timing again is arbitrary whereas 24 hours is a convenient time point to make this discrimination. For clinical samples an approved laboratory is required to follow a test SOP to report all data which is used in patient care. Thus the two assays you are inquiring about have been standardized, in part for convenience, but also for accuracy and reproducibility of results. These SOP's have been agreed upon between many laboratories and regulatory authorities in an attempt to attain comparable inter and intra lab data. This, of course, has spilled into the research arena as "The acceptable procedure to follow"
Hemolysis occurs very quickly because of disruption of membrane functions that is one part of toxicity to the cell. As a whole cytotoxicity is related to three different aspects 1. toxicity of any toxicant to biomolecules such as enzymes, proteins, vitamins, lipids, factors, cytokines, complements and growth factors basically related to inhibition or acceleration of activity. 2. Effect of toxicants on cell organelles 3. Effect of toxicant on cytochromes, genes and alteration of gene expression and last is the death or lethality. Cytotoxicity deals with morbidity and mortlaity, while hemolysis is a simple toxic effect, though it comes under cytotoxicity.
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